Process and apparatus for the preparation of chlortetracycline-containing animal feed compositions

ABSTRACT

The present invention provides improved chlortetracycline-containing animal feed compositions and processes and apparatuses for their preparation. In certain embodiments, raw fermentation broth comprising chlortetracycline is divided into two portions. The first portion is mixed with a compound that complexes chlortetracycline. The second portion is acidified and the solids are removed. The acidified liquid is treated with a complexing agent to produced a chlortetracycline complex. The first and second portions thus treated are then mixed and the mixture is passed on to a filter press or other means for separation of the solids to produce a wet cake comprising complexed chlortetracycline. In alternative embodiments, the second portion may be acidified and filtered and admixed with the first portion prior to the complexing step. The resulting mixture is passed on to a filter press or other means for separation of the solids. In still further embodiments, the complexing steps are replaced with neutralizing or basification steps to yield the free base form of chlortetracycline. The wet filter cake comprising chlortetracycline and fermentation solids is dried and sized to produce a dried semi-finished product in the form of a powder, meal, granules, pellets, tablets, etc. The semi-finished product may be standardized to produce an animal feed supplement having a desired potency. In a further aspect, the present invention relates to a method of combating microbial infection and promoting growth in animals comprising orally administering to said animals an effective amount of an animal composition according to the present invention.

INCORPORATION BY REFERENCE

The present application claims Priority as a Continuation-in-Part under35 USC Section 119(e) to provisional U.S. application No. 60/213,424,filed Jun. 22, 2000, and claims priority under 35 USC Section 120 toU.S. application Ser. No. 09/350,474, filed Jul. 9, 1999, now U.S. Pat.No. 6,761,899 U.S. application Ser. No. 09/587,410, filed Jun. 5, 2000,now U.S. Pat. No. 6,562,615 and U.S. application Ser. No. 09/587,411,filed June 5, 2000, now U.S. Pat. No. 6,506,402.

Further, each of the following applications are incorporated herein byreference in its entirety: provisional U.S. application Nos. 60/213,424filed Jun. 22, 2000, 60/103,074 filed Oct. 5, 1998, 60/105,229, filedOct. 21, 1998, 60/115,761, filed Jan. 13, 1999, and U.S. applicationSer. No. 09/350,474, filed Jul. 9, 1999, and U.S. application Ser. No.09/587,410, filed Jun. 5, 2000, and U.S. application Ser. No.09/587,411, filed Jun. 5, 2000.

FIELD OF THE INVENTION

The present invention relates to an improved process for the preparationof chlortetracycline-containing compositions suitable for incorporationin animal feed.

BACKGROUND OF THE INVENTION

Antibiotics, such as tetracyclines, are used as growth promoters andfeed efficiency promoters in animals such as poultry and livestock, andfor therapeutic and prophylactic disease control in animals such aspoultry and livestock, fish, domesticated pets, and so forth. Suchantibiotics are typically formulated in an animal feed premix or animalfeed supplement containing the antibiotic and an edible carrier ordiluent. These premixes or animal feed supplements may then be mixedwith a sufficient quantity of an appropriate animal feed (e.g.,livestock, poultry, fish, pet and/or other animal feed) to provide afinal animal feed formulation having the desired level of activecompound in the feed.

Prior art chlortetracycline-containing animal feed supplements preparedthrough normal fermentation and dehydration of the mycelium mealprovide, at maximum, chlortetracycline potencies of about 26% to 28% byweight. The highest concentration currently available in a standardizedfeed supplement is 22%. (Since chlortetracycline hydrochloride is thereference standard for chlortetracycline feed grade materials, allchlortetracycline concentrations and percentages stated herein, unlessindicated otherwise, are calculated as the hydrochloride equivalent,regardless of the form present (e.g., the free base, complexes or saltsother than the hydrochloride, etc.)). In order to obtain higherchlortetracycline concentrations, it is possible add pure crystallinechlortetracycline hydrochloride USP to the mycelium meal to raise thepotency. This, however, is not economical. Furthermore, becausechlortetracycline hydrochloride is water soluble, there is a question ofstability in finished feeds containing moistures of 10% or higher.

It would therefore be desirable to provide achlortetracycline-containing animal feed premix or animal feedsupplement suitable for use in the veterinary, animal nutrition, andanimal husbandry arts that can be prepared directly from a fermentationproduct and that can be prepared in a high concentration withoutrequiring the addition of the antibiotic in pure form and that hasimproved stability over chlortetracycline hydrochloride feedsupplements.

SUMMARY OF THE INVENTION

The present invention provides an improved process and apparatus for thepreparation of a fermentation cake or mycelium meal material containingchlortetracycline. The present invention also relates to thechlortetracycline-containing fermentation cake or mycelium meal materialso produced and chlortetracycline-containing animal feed additivesderived therefrom. It has been discovered that the procedures inaccordance with this teaching produce chlortetracycline-containingfermentation products having higher concentrations than possible fromprior fermentation/dehydration processes, thus making it possible toproduce animal feed supplements which take up less room in feedcomplexes, require less storage space, reduce packaging and shippingcosts, and so forth. The present invention also relates to animal feedcompositions prepared in accordance with this teaching.

In certain embodiments, the present invention provideschlortetracycline-containing animal feed additives having achlortetracycline concentration of about 35% or greater. In preferredembodiments, the present invention provides chlortetracycline-containinganimal feed additives having a chlortetracycline concentration of about40% to about 70%, or greater. In more preferred embodiments, the presentinvention provides chlortetracycline-containing animal feed additiveshaving a chlortetracycline concentration of about 44% to about 55%.

In additional embodiments, the present invention also relates to methodsfor preventing or combating microbial infection in animals comprisingorally administering to said animals a prophylactic or therapeuticamount of an animal comestible composition comprising a medicated feedsupplement according to the present invention. In yet a furtherembodiment, the present invention relates to methods for increasing feedefficiency, promoting animal growth, and increasing rate of weight gainand in an animal comprising orally administering to said animals aneffective amount of an animal comestible composition comprising amedicated feed supplement according to the present invention.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed. The accompanyingdrawings, which are incorporated in and constitute a part of thespecification, illustrate various embodiments of the invention andtogether with the general description, serve to explain the principlesof the invention.

BRIEF DESCRIPTION OF THE DRAWING

The numerous objects and advantages of the present invention may bebetter understood by those skilled in the art by reference to theaccompanying figures in which:

FIGS. 1 and 2 are flow charts outlining processes for preparing achlortetracycline-containing animal feed composition in accordance withtwo embodiments of the present invention wherein the chlortetracyclineis present in the form of a complex;

FIGS. 3 and 4 are flow charts outlining processes for preparing achlortetracycline-containing animal feed composition in accordance withtwo embodiments of the present invention wherein the chlortetracyclineis present in the form of the free base; and

FIGS. 5 and 6 are schematic diagrams of two systems designed forcarrying out procedures of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The chlortetracycline animal feed supplements in accordance with thepresent invention are prepared by fermentation of a microbial sourceorganism that produces chlortetracycline, including but not limited toStreptomyces aureofaciens, as is generally known in the art. Thefermentation process may be any suitable fermentation process, and maybe a commercial or industrial scale fermentation process. Generally, aninoculum is started by collecting the spores of a chlortetracyclineproducing organism such as Streptomyces aureofaciens or any otherchlortetracycline producing species which are germinated to produce anactively growing culture of the microbial source organism. A sterilized,e.g., autoclaved, agar slant or other slant medium is inoculated withthe actively growing culture and incubated. The spores produced are thenplaced into a small amount of liquid, e.g., distilled water, nutrientmedium, etc., to produce an aqueous spore suspension which is propagatedto produce the inoculum for the fermentation operation. The resultinginoculum is introduced into a fermentation medium to produce afermentation broth comprising the fermentation medium and inoculumwhereby the chlortetracycline is produced by fermentation in thefermentation broth. In one embodiment, an inoculum may be prepared in aninsemination fermentor in a suitable nutrient medium, and the inoculumthus prepared may be transferred to an industrial fermentor.

Cultivation of Streptomyces aureofaciens or other source organism may becarried out in a wide variety of liquid media. Media which are usefulfor the production chlortetracycline include an assimilable source ofcarbon and an assimilable source of nitrogen. Assimilable ormetabolizable sources of carbon and nitrogen are generally known in theart. Exemplary of sources of carbon include, for example, glucose orother sugars, including sucrose, maltose, dextrose, and the like,polysaccharide, starches such as corn starch, potato starch or the like,molasses, glycerol, sugar alcohols, oil and fats, and so forth.Exemplary sources of nitrogen include, for example, protein, proteinhydrolysates (e.g., peptones), polypeptides, amino acids, corn steepliquor, beef or other meat extracts, soy protein products (e.g.,isolates, flours, meals, etc.) yeast protein (e.g., yeast extracts,yeast autolysates, etc.), casein (hydrolyzed or unhydrolyzed), fishmeal, urea, nitrates, ammonia, and various other nitrogenous substancesof plant or animal origin. Other ingredients may be supplied asnecessary, and in many cases may be present in the nitrogen source orother constituents of the media, including enzymes, complex growthfactors (e.g., vitamins), inorganic anions and cations, such aspotassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate,chloride, etc., and trace elements such as boron, molybdenum, copper,cobalt, iron, copper, zinc, etc.

Oxygen is supplied to the medium during fermentation. Aeration isprovided by forcing sterile air through or onto the surface of thefermenting medium.

Although aeration alone may provide the desired agitation, furtheragitation is generally desirable, e.g., as provided by a mechanicalimpeller located within the vessel. An anti-foaming agent such as avegetable oil, lard oil, silicone defoamer, hydrocarbon petroleum, andthe like may be added to control foaming as needed.

The fermentation medium is brought to a temperature of about 25° C. toabout 40° C., preferably about 28° C. to about 35° C. The length of timethat the fermentation process is allowed to continue depends on the anumber of factors, including the composition of the fermentation medium,temperature, quantity of cells in the inoculum, quantity and/orconcentration of chlortetracycline desired in the fermentation broth,and the ability to avoid contamination of the fermentation medium withother cultures. Typically, for batch-wise production, the fermentationtime can vary from about 50 hours to about 200 hours.

It will be recognized that the present invention may also be implementedusing a continuous fermentation process. After chlortetracyclineproduction achieves a steady state, chlortetracycline production may bemaintained by the continuous addition of fermentation medium (e.g.,without addition of new inoculum) and continuous removal of fermentationbroth at a rate such that the volume of fermentation broth in thefermentation tank and the concentration of chlortetracycline in thefermentation broth remain essentially constant. Alternatively, theremoval and addition may be performed in intermittent fashion ratherthan continuously so long as the average broth volume remainsessentially constant. The production rate may be adjusted by control ofthe medium composition and other fermentation conditions, as well as therate of addition and removal, so as to optimize or provide a desiredrate of chlortetracycline production. In theory, continuouschlortetracycline production can continue indefinitely, however, inpractice, it is limited by contamination of the fermentation medium withother microorganisms, equipment malfunction, abnormalities in theprocess, and so forth. Continuous production may be continued for up toseveral weeks or longer, or until chlortetracycline production decreasesto an uneconomical level.

The certain embodiments, an appropriate amount of fermentation mediumcontaining the entire amount of carbon and nitrogen source material tobe consumed during the fermentation process may be added to thefermentation tank at the beginning of the fermentation process. However,it has been found in accordance with this teaching that the furtheraddition of nitrogen source materials after fermentation has begunincreases potency by 5% or more. The additional nitrogen source may beadded by a number of methods. In certain embodiments, an appropriateamount of nitrogen source material may be initially added to thefermentation medium and addition is continued after the fermentation hasbegun. In certain embodiments, a single quantity of nitrogen sourcematerial may be added after a predetermined period of time, such as 12,24, 36, 48, etc., hours after the fermentation is begun, or when thenitrogen level in the fermentation medium drops below some predeterminedlevel. In other embodiments, the additional nitrogen source is added asan intermittent series of additions, for example, at predetermined timeintervals, or when the nitrogen level in the fermentation medium dropsbelow some predetermined level. For example, in preferred embodiments,relatively small, frequent additions of a nitrogen source material aremade. The additions are in an amount sufficient to maintain the nitrogenlevel within a desired range. The frequency may be, for example,half-hourly (or more frequently) to about bihourly, preferably hourly.In still other embodiments, the additional nitrogen source is addedcontinuously, beginning at the time the fermentation is begun, at someperiod of time after the fermentation is begun, or when the nitrogenlevel in the fermentation medium drops below some predetermined level.The additional nitrogen source material is added to the fermentor insuch a quantity and/or rate that will maintain the nitrogenconcentration at or above the minimum level required for fermentation.Typically, the additional nitrogen is added so that the nitrogenconcentration is maintained at about 30-300 mg/100 mL, more typicallyabout 60-120 mg/100 mL.

Additional carbon source material may also be added during thefermentation operation. Similarly, a single quantity of carbon sourcematerial may be added after a predetermined period of time, such as 12,24, 36, 48, etc., hours after the fermentation is begun, or when thecarbon level in the fermentation medium drops below some predeterminedlevel. In other embodiments, the additional carbon source is added as anintermittent series of additions, for example, at predetermined timeintervals, or when the carbon level in the fermentation medium dropsbelow some predetermined level. For example, in preferred embodiments,relatively small, frequent additions of a carbon source material aremade. The additions are in an amount sufficient to maintain theassimilable carbon level within a desired range. The frequency may be,for example, half-hourly (or more frequently) to about bihourly,preferably hourly. In still other embodiments, the additional carbonsource is added continuously, beginning at the time the fermentation isbegun, at some period of time after the fermentation is begun, or whenthe carbon level in the fermentation medium drops below somepredetermined level. The additional carbon source material is added tothe fermentor in such a quantity and/or rate that will maintain thecarbon concentration at or above the minimum level required forfermentation. Typically, the additional carbon source or sources areadded so that the amount of carbon source material in the fermentationmedium concentration is maintained at about 0.1% to about 10% by weight,more typically about 0.5% to about 5% by weight. In a preferredembodiment wherein an economical method is desired nutrient supplementsmay be added at levels and rates determined after testing wherein apreferred rate of about six (6) hours has been beneficially employed.

When the concentration of chlortetracycline reaches the desired level inthe fermentation broth, the fermentation broth, including the biomassand other fermentation solids, is harvested and divided into twoportions. It is not necessary that a single fermentation batch bedivided into two portions and alternatively, the two portions may beharvested from two or more fermentation tanks. Likewise, the twoportions may be derived separate batches of a single fermentor whereinone portion is derived from a first fermentation batch and the secondportion is derived from a subsequent fermentation batch. For the sake ofbrevity, the present invention will be shown and described hereinprimarily by way of reference to harvesting the fermentation broth froma single fermentation tank and dividing the harvested broth into twoportions, however, it will be recognized that such teachings areintended to encompass and are equally applicable to obtaining the firstand second portions from a plurality of fermentation tanks or aplurality of fermentation batches obtained from a single fermentor.

The first portion of the harvested fermentation broth is sent to amixing tank. If the finished feed supplement is to containchlortetracycline as the free base, the harvested fermentation broth maybe treated with a base, such as ammonia, in an amount to neutralize thechlortetracycline. In forming the free base form of chlortetracycline,any base known to those skilled in the art can be used to neutralize thechlortetracycline-containing to form the free base form ofchlortetracycline. Preferably, the base is an aqueous ammonia solution.Other exemplary bases include but are not limited to alkali metalhydroxides, preferably aqueous solutions thereof, such as aqueous sodiumhydroxide. If the finished feed supplement is to contain complexedchlortetracycline, the harvested fermentation broth is admixed with acompound that complexes chlortetracycline, preferably in an amountsufficient to completely complex the tetracycline.

The second portion of the fermentation broth is sent to an acidificationtank for treatment with an acid and dilution. The acidified broth isthen passed on for separation of the solids. The separation may beperformed in a number of ways, including filtration, centrifuging,siphoning, decanting, pumping off, evaporation, and so forth. In oneembodiment, the filtration is carried out in a filter press or rotaryvacuum filter. In a preferred embodiment, the fermentations solids areseparated by pumping the fermentation broth through a filter press orwith a centrifuge. The filtrate is optionally recycled to the filter fora second filtration.

The filtrate containing the chlortetracycline acid addition salt ispassed on to a precipitation tank for precipitating thechlortetracycline. If the finished feed supplement is to containchlortetracycline as the free base, precipitation is performed byneutralizing the chlortetracycline acid addition salt to form thechlortetracycline free base through addition of a sufficient quantity ofa base such as ammonia or other base. Alternatively, if the finishedfeed supplement is to contain complexed chlortetracycline, precipitationis performed by adding a complexing agent, preferably in an amountsubstantially sufficient to completely complex the chlortetracycline.

Where the finished feed supplement contains complexed chlortetracycline,suitable complexing agents are those having the ability to complex withthe chlortetracycline at stable pH ranges. Exemplary complexing agentsinclude but are not limited to salts and bases of calcium, magnesium,aluminum, iron, manganese, zinc, and copper, preferably metal saltswhich exhibit alkaline behavior in aqueous media, e.g., salts of weakacids. Preferred complexing agents include alkaline earth metalcarbonates such as magnesium carbonate and calcium carbonate, basicalkaline earth metal compounds such as calcium hydroxide, magnesiumhydroxide, calcium chloride, magnesium chloride, calcium oxide,magnesium oxide, calcium sulfate, magnesium sulfate, calcium phosphate,magnesium phosphate, and so forth. The preferred complexing compound iscalcium carbonate. The amount of complexing agent needed can be readilydetermined by those skilled in the art.

The first and second portions, treated as described above, are thenmixed and this mixture is then passed for separation of the solids.Separation of the solids, such as filtration, centrifugation,evaporation, or other solid-liquid separation technique, yields a wetfermentation cake comprising chlortetracycline (either as the free baseor as a complex), the fermentation biomass and other fermentationsolids.

It is not necessary that the first and second portions be separatelyprecipitated prior to admixture. In an alternative embodiment theharvested raw fermentation broth is again divided into first and secondportions. The first portion is left in its raw state and the secondportion of the fermentation broth is sent to an acidification tank fortreatment with an acid as described above. The acidified broth is thenpassed on for separation of the solids as described above.

The acidified filtrate containing the chlortetracycline activity fromthe second portion is then mixed with the first portion of thefermentation broth in its raw state.

If the finished feed supplement is to contain chlortetracycline as thefree base, the resulting mixture is treated with a base, such as ammoniaor other base, in an amount sufficient to neutralize thechlortetracycline. If the finished feed supplement is to containcomplexed chlortetracycline, the harvested fermentation broth is admixedwith a compound that complexes chlortetracycline as detailed above,preferably in an amount sufficient to completely complex thetetracycline. The mixture containing precipitated chlortetracycline(free base or complex) is then passed for separation of the solids asdescribed above.

The wet filter cake (biomass) produced in accordance with the presentinvention containing precipitated chlortetracycline is then dried andsized. The drying step may be performed by any conventional technique,which may involve the use of any suitable drying means, such as an oven,tray dryer, tunnel dryer, spray dryer, spray granulator, fluid beddryer, shelf dryer, drum dryer, rotary dryer, microwave dryer, contactdryer, and the like.

The wet cake may optionally be subjected to a compression or compactionstep, e.g., to produce pellets, tablets, sheets, ribbons, briquets,extrudates of a desired cross-sectional size and shape, etc., which mayoptionally be subjected to further size reduction as needed. Theoptional compaction step may involve the use of any suitable compactingor compressing means, such as a pellet press or pellet mill, punch anddie tableting equipment, compression or compaction rollers, extruders,and the like. The compaction or compression step may be performed priorto drying or at an intermediate drying stage, (e.g., paste, slurry,etc.), or after drying. Where a compaction or compression step is used,there may optionally be added one or more fillers, binders, humectants,disintegrating agents, solution retarding agents, absorptionaccelerators, wetting agents, absorbents, lubricants, or otherexcipients or carriers as would be known to those skilled theveterinary, animal nutrition, and animal husbandry arts.

The sizing step may be performed by any conventional technique toproduce a chlortetracycline-containing semi-finished product having adesirable particle size or range of particle sizes. The desired particlesize of the semi-finished product will generally depend on the intendedapplication for the feed supplement. The particle size may range fromfinely divided powders to relatively coarse particles or granules, forexample, having a diameter of up to about 5 mm or larger. Such formsinclude powder, meal, pellet, granule, tablet, or other similar forms.In certain embodiments, a drying method may be selected that gives thedesired particle size (for example, spray drying to a powder) or thatgives oversized particles that can be further reduced in size (forexample, tray drying the filter cake in tact or in relatively largepieces). Oversize particles may then be further reduced in size togranules or a powder by grinding, milling, screening, pulverizing,crushing, machining, and so forth.

In certain other embodiments, the selected drying method may produceparticles that are smaller than desired, in which case the smallparticles can be built up into larger particles, for example, via anagglomeration process, or via a compaction or compression process asdescribed above. For example, a powder (e.g., produced by spray dryingor by mechanically reducing larger particles) may be made into pelletsor granules by agglomeration. Alternatively, undersized particles may becompacted, compressed, extruded, etc., as described above.

The sized particles may be subjected to further drying stages aftersizing, for example, where the sizing step involves introduction ofwater (e.g., agglomeration using water) or where the particles are sizedbefore the desired degree of dryness is achieved.

A drying and/or sizing process may produce a distribution of particlesizes wherein only a portion of the particles are within the desiredrange of particle sizes. In such cases, the oversized and undersizedparticles may be removed, e.g., by screening. The oversized particlesmay be further reduced in size as described above and undersizedparticles may be built up into larger particles via agglomeration,compression or compaction as described above. Alternatively, theundersized particles may be screened and used to increase thechlortetracycline concentration in subsequent harvested fermentationbatches.

In certain embodiments, the dry cake is then sized by grinding, milling,pulverizing, screening, etc., to provide the complexedchlortetracycline-containing fermentation solids in particulate form.The particles are screened to produce a semi-finished product having adesired particle size and it has been discovered that the particles soproduced provide a solid particulate feed material having good strengthand fracture toughness without compression or compaction of theparticles.

After drying and sizing, the semi-finished product containingchlortetracycline complex or free base may optionally be standardized toproduce a finished product having a desired potency by adding one ormore of a diluent, such as one or more of an edible feed material, amineral product, and an oil.

The term “semi-finished product” is used herein to refer to the productresulting from drying and sizing the filter cake and the term “finishedproduct” is used herein to refer to a product resulting from admixtureof the semi-finished product and one or more diluents (e.g., one or moreof an edible feed material, a mineral product, and an oil) to provide ananimal feed premix having a predetermined or standardized concentrationof the active ingredient. It will be recognized, however, that thesemi-finished product itself, once assayed for chlortetracyclineactivity, is itself also suitable for use as an animal feed premix.

The terms “animal feed premix,” “animal feed supplement,” and “animalfeed additive” are generally used herein interchangeably, withoutattention to any nuances in meaning, to mean a concentrated additivepremix comprising chlortetracycline which may be added to an animal'sfeed to form a medicated comestible composition in accordance with thepresent invention. The terms “final feed mixture” or “final feedproduct” will generally be used herein to mean an animal comestiblecomprising an animal's feed and a medicated animal feed supplementaccording to the present invention.

The animal feed supplements according to the present invention may beblended with an animal feed to produce a medicated finished complete orsupplement feed product. Animals to which the feed supplements accordingto the present invention may be administered include pets or companionanimals, and ranch or farm animals or other livestock, such as animalsraised as a food source or other commercial purpose. Such animalsinclude, but are not limited to, cattle, sheep, horses, pigs, buffalo,goats, dogs, cats, rabbits, rats, mice, minks, fish, and fowl (includingegg-laying or edible fowl, such as chickens, turkeys, geese, ducks,quail, pheasant, etc.), and so forth.

The medicated animal final feed mixtures according to the presentinvention are prepared by adding an animal feed premix prepared inaccordance with the present invention to an animal's food. The premixmay be added to the food in a number of ways. The feed premix containinga given quantity of chlortetracycline may be added to a given quantityof feed and mixed or blended to provide a substantially homogeneousmedicated feed composition. Large feed lots may be prepared in thismanner for treating a large number of animals. Alternatively, feedbatches containing feed for a single animal or single meal may beprepared either by mixing a predetermined quantity of premix accordingto the present invention with the animal feed or by adding apredetermined quantity of premix to an animal's feed as a top dressing.

By varying the quantity of feed supplement added to the feed, theconcentration of the active ingredient in the final feed mixture may beadjusted to meet particular needs and may be varied over a wide range.The minimum concentration should be such as to achieve the desiredresult (therapeutic, prophylactic, growth enhancement, etc.). Themaximum concentration should be such as to avoid any undesirable sideeffects when the feed rations are ingested by the animal. Within theselimitations, specific amounts of active ingredient will normally beregulated by the practitioner according to potency of the premix and theusual recommended dosing levels for the active ingredient. Thepractitioner will generally take into account a number of factors, suchas the animal species being treated, the animal's age or stage ofdevelopment, the frequency of administration, whether the composition isbeing administered therapeutically or prophylactically and the degree ofantimicrobial results sought, the severity of any disease being treated,and so forth. The selection of dosages and dosage regimens is generallyperformed as a routine matter by persons skilled in the arts pertainingto veterinary medicine, animal husbandry and nutrition, and the like.Accordingly, the indications and dosage levels given herein are intendedto be exemplary and illustrative only, and are not intended to beexhaustive or restrictive of the present invention as claimed.

In a preferred aspect, the present invention provides a semi-finishedproduct having a concentration higher than can be achieved throughnormal fermentation and dehydration means. In certain embodiments, thesemi-finished product has a chlortetracycline concentration of about 35%or greater. In preferred embodiments, the present invention providessemi-finished product having a chlortetracycline concentration of about40% to about 80%, or greater. In more preferred embodiments, the presentinvention provides chlortetracycline-containing animal feed additiveshaving a chlortetracycline concentration of about 50% to about 70%.

The semi-finished product in accordance with the present invention maybe standardized as described above to provide an animal feed supplementhaving a desired or predetermined activity. In certain embodiments, thepresent invention provides chlortetracycline-containing animal feedadditives having a chlortetracycline concentration of about 35% orgreater. In preferred embodiments, the present invention provideschlortetracycline-containing animal feed additives having achlortetracycline concentration of about 40% to about 60%, or greater.In more preferred embodiments, the present invention provideschlortetracycline-containing animal feed additives having achlortetracycline concentration of about 44% to about 55%.

In some of the embodiments according to the present invention, theanimal feed supplements according to the present invention will containa chlortetracycline concentration ranging from about 10 g/lb to about300 g/lb, and preferably from about 90 g/lb to about 200 g/lb.

Thus, in formulating the final animal comestible feed comprising theanimal feed premix according to the present invention to contain theproper amount of active ingredient, one may calculate the amount ofactive ingredient it is desired to it administer to each animal, takeinto account the amount of feed per day normally consumed by the animal,and compute the proper concentration of chlortetracycline needed in thefeed, and add the appropriate amount of animal premix to achieve theproper concentration of chlortetracycline in the final feed mixture.

In some of the preferred embodiments according to the present invention,the final feed comprising an animal feed and the animal feed supplementin accordance with the present invention will generally containchlortetracycline in an amount of about 0.1 g/ton or greater, generallyranging from about from about 10 g/ton to about 3000 g/ton, preferablyfrom about 50 g/ton to about 2000 g/ton, and more preferably, from about50 g/ton to about 800 g/ton. Some preferred dosages for various animalsand indications are given below.

Animal Indications for Use Dosage Chickens For an increased rate ofweight gain and improved 10-50 feed efficiency g/ton Chickens Forcontrol of infectious synovitis caused by 100-200 Mycoplasma synoviae ¹g/ton Chickens For control of chronic respiratory disease (CRD) 200-400and air sac infection caused by Mycoplasma g/ton gallisepticum andEscherichia coli ^(1,2,3) Chickens For reduction of mortality due toEscherichia coli 500 g/ton infections^(2,4,5) Turkeys For an increasedrate of weight gain and improved 10-50 feed efficiency g/ton Turkeys Forcontrol of infectious synovitis caused by 200 g/ton Mycoplasma synoviae¹ Turkeys For control of hexamitiasis caused by Hexamita 400 g/tonmeleagrides ¹ Turkeys In turkey poults not over 4 weeks of age, for 400g/ton reduction of mortality due to paratyphoid caused by Salmonellatyphimurium Turkeys For control of complicating bacterial organisms 25mg/lb associated with bluecomb (transmissible enteritis, bodycoronaviral enteritis)^(1,2,3) wt/day Sheep For an increased rate ofweight gain and improved 20-50 feed efficiency g/ton Sheep For reducingthe incidence of (vibrionic) abortion 80 mg/ caused by Campylobacterfetus infection in head/day breeding sheep³ Swine For an increased rateof weight gain and improved 10-50 feed efficiency g/ton Swine Forreducing the incidence of cervical 50-100 lymphadenitis (jowl abscesses)caused by Group g/ton E. Streptococci Swine In breeding swine, forcontrol of leptospirosis 400 g/ton (reducing the instances of abortionand shedding of leptospirae) caused by Leptospira pomona ⁶ Swine Fortreatment of bacterial enteritis caused by 10 mg/lb Escherichia coli andSalmonella choleraesuis and body bacterial pneumonia caused by causedPasteurella wt/day multocida ^(3,7) Calves In calves up to 250 pounds,for an increased rate 0.1 mg/lb of weight gain and improved feedefficiency⁸ body wt/day Calves In calves 250-400 pounds, for anincreased rate 25-70 mg/ of weight gain and improved feed efficiency⁸head/day Cattle In growing cattle (over 400 pounds), for an 70 mg/increased rate of weight gain and improved feed head/day efficiency andreduction of liver condemnation due to liver abscesses³ Cattle For thecontrol of bacterial pneumonia associated 350 mg/ with shipping fevercomplex head/day Cattle In beef cattle under 700 lbs, for control ofactive 350 mg/ infection of anaplasmosis caused by Anaplasma head/daymarginale Cattle In beef cattle over 700 lbs, for control of active 0.5mg/lb infection of anaplasmosis caused by Anaplasma body marginale⁵wt/day Cattle In calves⁸, beef, and non-lactating dairy cattle, for 10mg/lb treatment of bacterial enteritis caused by body Escherichia coliand bacterial pneumonia caused wt/day by Pasteurella multocidaorganisms^(5,9) ¹Feed continuously for 7 to 14 days ²Do not feed topoultry producing eggs for human consumption ³Zero-day withdrawal period⁴Feed for 5 days ⁵Withdraw 24 hours prior to slaughter ⁶Feedcontinuously for 14 days ⁷Feed for not more than 14 days ⁸A withdrawalperiod has not been established for this product in pre-ruminatingcalves ⁹Treat for not more than 5 days

The following Examples are intended to illustrate but not to limit theinvention. A flow chart outlining a process in accordance with thepresent invention illustrated by Examples 1-16 is shown in FIG. 1. Aflow chart outlining a process in accordance with the present inventionillustrated by Examples 1-7, 8A-12A, and 14-16 is shown in FIG. 2. Aflow chart outlining a process in accordance with the present inventionillustrated by Examples 1-8, 9B-13B, 14C-15C, and 16 is shown in FIG. 3.A flow chart outlining a process in accordance with the presentinvention illustrated by Examples 1-7, 8C-15C and 16 is shown in FIG. 4.

EXAMPLE 1 Raw Materials and Media Composition

A test tube is used to store the spore of the Streptomyces aureofacienschlortetracycline strain. Washed sand is screened through an 80 meshsieve. The sand is placed into a glass tube and sterilized at 121° C.,0.104 MPa for 30 minutes. After sterilization, the sand is dried andcooled. The strain spore of chlortetracycline is inoculated into asterilized tube containing slant media using an inoculation needle underaseptic conditions. The tube is sealed with a cotton ball. Theinoculated media is cultivated for 4-5 days at 30 to 40° C., preferably35°±1° C., under thermostatically controlled conditions. The media iscooled and kept in the refrigerator at 4° C. until ready for use.

EXAMPLE 2 Slant Media

The composition of the culture media is as follows:

Ingredient Preferred Material Preferred Range (%) Carbon Source WheatBran 3.0-3.5 Activator MgSO₄ 0.004-0.006 Nitrogen Source (NH₄)₂HPO₄0.01-0.02 Activator K₂HPO₄ 0.008-0.012 Solidifier Agar 0.05-0.10Moisturizer Water Remainder

A single colony is selected from cultured media described above underaseptic conditions and inoculated on a slant media as described above.The inoculum is then cultivated for 4-5 days at 30 to 40° C., preferably35°±1° C., under thermostatically controlled conditions. After thegrowth of the spore, the well-grown slant is selected and kept in arefrigerator at between 3° C. to 5° C. until ready for use.

EXAMPLE 3 Seed Media

The composition of the seed media is as follows:

Ingredient Preferred Material Preferred Range (%) Carbon Source CornStarch 3.0-4.0 Nitrogen Source Peanut Meal or Soybean 1.5-3.5 MealNitrogen Source Peptone 0.4-0.6 Nitrogen Source Yeast Powder 0.4-0.8Nitrogen Source (NH₄)₂SO₄ 0.2-0.4 Carbon Source & Buffer CaCO₃ 0.2-0.4Activator NaCl 0.1-0.3 Activator KH₂PO₄ 0.02-0.04 Defoamer Vegetable Oil0.3-0.4 Moisturizer Water 90.0-93.9

The seed culture media is prepared in a container tank and stirred untilcompletely homogeneous. The media is fed into a seed tank through asieve. Water is added and the tank is closed. The tank is sterilizedwith steam and cooled. The seed media is inoculated and aeration andagitation is started. The air flow rate is from 0.5 to 2.0 m³ per minuteper m³ fermentation broth. The temperature is held at 25° C. to 35° C.for 24 to 36 hours. The pH should be about 6.0, the amino nitrogencontent should be from 60-120 mg/100 mL, and chlortetracycline potencyshould be greater than 75 u/mL.

EXAMPLE 4 Fermentation Media

The composition of the media for fermentation tanks will consists of thefollowing ingredients and respective ranges. The media is prepared andtransferred to the fermentation tank.

Ingredient Preferred Material Preferred Range (%) Carbon Source CornStarch 7.0-9.0 Nitrogen Source Peanut Meal or Soybean 3.0-3.5 MealNitrogen Source Peptone 0.4-0.6 Nitrogen Source Yeast Powder 0.4-0.8Bio-Catalyst α-amylase 0.01-0.02 Nitrogen Source (NH₄)₂SO₄ 0.5-1.0Carbon Source & Buffer CaCO₃ 0.4-0.7 Activator KH₂PO₄ 0.02-0.03Activator NaCl 0.1-0.3 Activator MgSO₄ 0.01-0.03 Nitrogen SourceCorn-Steep Liquor 1.2-1.8 Defoamer Vegetable Oil 0.15-.025 MoisturizerWater 86.8-82.0

EXAMPLE 5 Sugar Tank Composition

The sugar preparation added to the fermentation tank during fermentationis composed of the following:

Ingredient Preferred Material Preferred Range (%) Carbon Source Starch40-50 Nitrogen Source Peanut Meal or Soybean   0-0.6 Meal NitrogenSource Corn-Steep Liquor 1.0-2.5 Bio-Catalyst α-amylase 0.05-0.08 CarbonSource & Buffer CaCO₃ 0.4-0.8 Activator NaCl 0.2-0.4 Nitrogen SourcePeptone   0-0.3 Nitrogen Source Yeast Powder   0-0.3 Activator CaCl₂  0-0.3 Defoamer Vegetable Oil 0.15-.025 Moisturizer Water 44.5-58.2

EXAMPLE 6 Air Used During the Production Process

Air used during the fermentation process should be sterilized prior tobe being injected to the fermentation or other media. The air is drawninto a compressor through a filter consisting of metallic net and foamrubber. Any impurities in the air, such as dirt, dust, sand, etc., arethen removed. The air from the compressor should be cooled to about 15°C. to about 35° C. The cooled air is then ridded of oil and water bypassing through an oil and water separator. The air is then heated toabout 40° C. to about 50° C. The air is then passed through a filtrationsystem. The filtered air is then fed to the seed and fermentor tanks foruse in aeration in the fermentation process.

EXAMPLE 7 Fermentation

The fermentation culture media is pumped from the compounding pool intothe fermentor. The media is sterilized by the introduction of steam.After the fermentation media has cooled, the seed culture media ispumped into the fermentor through a sterile tube. Cultivation then takesplace. During fermentation, the temperature is maintained at 25° C. to40° C., preferably about 28° C. to about 35° C., for 120 to 140 hours,with sterilized sugar media added as required with aeration. Duringfermentation, ammonia (for example, aqueous ammonia, about 20% orgreater) is introduced into the fermentor as necessary to maintain pH at5.5 to 6.3.

The following EXAMPLES 8 through 13 illustrate a first embodiment ofharvesting the fermentation broth and recovering complexedchlortetracycline in accordance with the present invention.

EXAMPLE 8 Harvesting of Fermentation Broth

When the desired potency is reached, e.g., about 15,000 u/mg to about30,000 u/m, or higher where a higher potency is desired, preferablyabout 25,000 u/mg to about 29,000 u/mg, the fermentation process isended and the fermentation broth is discharged from the fermentationtank and divided into two portions. Discharge of the fermentation brothmay be carried out by any convenient technique, such as using a pump,pressure, gravity, etc. A first portion is sent directly to a complexingtank for complexing and precipitation of the calcium complex ofchlortetracycline. A second portion of the harvested broth is sent to anacidification tank for acidification and dilution. The ratio of thefirst portion of raw fermentation broth sent directly to the complexingtank for complexing and the second portion of the second portion of theraw fermentation broth removed for acidification preferably ranges fromabout 1:20 to about 20:1, and may be selected to give a desiredconcentration of chlortetracycline in the semi-finished product. In oneembodiment, the ratio of the first portion to the second portion rangesfrom about 1:10 to about 10:1. In another embodiment, the ratio of thefirst portion to the second portion ranges from about 1:5 to about 5:1.In still another embodiment, the ratio of the first portion to thesecond portion ranges from about 1:3 to about 3:1. In yet anotherembodiment, the ratio of the first portion to the second portion rangesfrom about 1:2 to about 2:1. In another embodiment, the ratio of thefirst portion to the second portion ranges from about 2:3 to about 3:2.In yet another embodiment, the ratio of the first portion to the secondportion is about 1:1.

EXAMPLE 9 Complexing First Portion of Raw Fermentation Broth

The first portion of the fermentation broth is sent to a complexingtank. The fermentation broth is optionally cooled, e.g., to below about20° C. Calcium carbonate (about 0.1% to about 4%, preferably about 0.4to about 1.0%, w/v, based on the volume of complexed fermentation broth)is added and aqueous ammonia or other suitable base is added to adjustthe pH to a value of about 7.0 or greater, preferably from about 7.0 toabout 9.0, and more preferably about 7.6 to about 7.8. The base ispreferably aqueous ammonia having a concentration of about 10% orgreater, preferably about 20% or greater.

EXAMPLE 10 Acidification of Second Portion of Raw Fermentation Broth

The second portion of the harvested broth is sent to an acidificationtank and cooled, preferably from about 15-20° C. The acid used may beany mineral acid or sufficiently strong organic acid as are known tothose skilled in the art. The acid is added in sufficient quantity toadjust the pH to about 4.0 or less, preferably about 3.0 or less, andmost preferably about 2.0 or less. In a preferred embodiment, the acidis an acid that forms a pharmaceutically acceptable salt ofchlortetracycline and most preferably is hydrochloric acid, hydrobromicacid, sulfuric acid, oxalic acid, or mixtures thereof. In an exemplaryembodiment, oxalic acid (2.0% calculated based on total quantity of theharvested broth), is added while agitating and HCl is added to adjustthe pH to about 1.1 to 1.3. Sodium ferrocyanide may be added in amountless than about 2.0% calculated based on total quantity of the harvestedbroth. In an exemplary embodiment, sodium ferrocyanide is added inamount of from about 0.1-0.2%, calculated based on total quantity of theharvested broth. Zinc sulfate may also be added in amount less thanabout 2.0% calculated based on total quantity of the harvested broth. Inan exemplary embodiment, zinc sulfate is added in amount of from about0.1-0.2%, calculated based on total quantity of the harvested broth. Anaqueous solution of oxalic acid (pH<about 3.0, preferably from about 1.5to about 1.8) is then added to dilute the acidified broth to obtainpotency at about 10,000 unit/ml.

EXAMPLE 11 Filtration of Acidified Fermentation Broth

The acidified broth is pumped to a filter press or other means forseparating the liquids. The filtrate containing the chlortetracyclinehydrochloride is collected and may be sent directly for complexing or,optionally, may be and recirculated through the filter press for asecond filtration prior to being passed on for complexing.

EXAMPLE 12 Complexing and Precipitation of Acidified Filtrate

The acidified filtrate is complexed in a manner similar to the firstportion.

Calcium carbonate (0.01 to about 10%, preferably about 0.1-2.0%, w/v,based on total volume of acidified broth) is added to the complexingtank containing the acidified broth. Aqueous ammonia or other suitablebase is added while agitating to adjust the pH to a value of about 7.0or greater, preferably about 7.0 to about 9.0, and more preferably about7.6 to about 7.8. The temperature is maintained at about 5 to about 15°C. The base is preferably aqueous ammonia having a concentration ofabout 10% or greater, preferably about 20% or greater. Stirring iscontinued for about 1 hour to obtain a suspension of chlortetracyclinecalcium complex.

EXAMPLE 13 Mixing

The complexed fermentation broth obtained from the first portion of theraw fermentation broth and the chlortetracycline calcium complexobtained from the acidified and filtered broth (second portion) aremixed and the mixture is cooled to about 20° C. or less. The mixture isstirred until well mixed, e.g., for about 3 to about 30 minutes,preferably for about 5 to about 10 minutes. After mixing, the mixture ispumped to a filter press or other suitable means for filtration andseparation.

The following EXAMPLES 8A through 12A illustrate a second embodiment ofharvesting the fermentation broth and recovering complexedchlortetracycline in accordance with the present invention.

EXAMPLE 8A Harvesting of Fermentation Broth

A chlortetracycline producing fermentation operation is performed asdescribed in Examples 1-7, above. When the desired potency is reached,e.g., about 15,000 u/mg to about 30,000 u/mg, or higher where a higherpotency is desired, preferably about 25,000 u/mg to about 29,000 u/mg,the fermentation process is ended and the fermentation broth isdischarged from the fermentation tank and divided into two portions.Discharge of the fermentation broth may be carried out by any convenienttechnique, such as using a pump, pressure, gravity, etc. The firstportion remains in its raw state and the second portion of the harvestedbroth is sent to an acidification tank for acidification. The firstportion is either discharged and stored until treatment (acidificationand filtration) of the second portion is completed, or, may remain inthe fermentor until treatment of the second portion is completed. Theratio of the first and second portions of the second portion of the rawfermentation broth preferably ranges from about 1:20 to about 20:1, andmay be selected to give a desired concentration of chlortetracycline inthe semi-finished product. In one embodiment, the ratio of the firstportion to the second portion ranges from about 1:10 to about 10:1. Inanother embodiment, the ratio of the first portion to the second portionranges from about 1:5 to about 5:1. In still another embodiment, theratio of the first portion to the second portion ranges from about 1:3to about 3:1. In yet another embodiment, the ratio of the first portionto the second portion ranges from about 1:2 to about 2:1. In anotherembodiment, the ratio of the first portion to the second portion rangesfrom about 2:3 to about 3:2. In yet another embodiment, the ratio of thefirst portion to the second portion is about 1:1.

EXAMPLE 9A Acidification of Second Portion of Raw Fermentation Broth

The second portion of the harvested broth (from Example 8A) is sent toan acidification tank and cooled, preferably from about 15-20° C. Theacid used may be any mineral acid or sufficiently strong organic acid asare known to those skilled in the art. The acid is added in sufficientquantity to adjust the pH to about 4.0 or less, preferably about 3.0 orless, and most preferably about 2.0 or less. In a preferred embodiment,the acid is an acid that forms a pharmaceutically acceptable salt ofchlortetracycline and most preferably is hydrochloric acid, sulfuricacid, and/or oxalic acid. In an exemplary embodiment, oxalic acid (2.0%calculated based on total quantity of the harvested broth), is addedwhile agitating and HCl is added to adjust the pH to about 1.1 to 1.3.Sodium ferrocyanide may be added in amount less than about 2.0%calculated based on total quantity of the harvested broth. In anexemplary embodiment, sodium ferrocyanide is added in amount of fromabout 0.1-0.2%, calculated based on total quantity of the harvestedbroth. Zinc sulfate may also be added in amount less than about 2.0%calculated based on total quantity of the harvested broth. In anexemplary embodiment, zinc sulfate is added in amount of from about0.1-0.2%, calculated based on total quantity of the harvested broth. Anaqueous solution of oxalic acid (pH<about 3.0, preferably from about 1.5to about 1.8) is then added to dilute the acidified broth to obtainpotency at about 10,000 unit/ml.

EXAMPLE 10A Filtration of Acidified Fermentation Broth

The acidified broth from Example 9A is pumped to a filter press or othermeans for separating the liquids. The filtrate containing thechlortetracycline hydrochloride is collected and may be sent directly tothe complexing tank or, optionally, may be and recirculated through thefilter press for a second filtration prior to being passed on to thecomplexing tank containing the first portion.

EXAMPLE 11A Mixing

The first portion of the raw fermentation broth (from Example 8A) andthe acidified and filtered broth containing chlortetracycline obtainedfrom the second portion (from Example 10A) are mixed, and the mixture isoptionally cooled to about 20° C. or less. The mixture is stirred untilwell mixed, e.g., for about 3 to about 30 minutes, preferably for about5 to about 10 minutes.

EXAMPLE 12A Complexing Mixture Containing the First Portion of RawFermentation Broth and Acidified Filtrate

The mixture of raw fermentation broth and acidified filtratefermentation from Example 11A broth is optionally cooled, e.g., to belowabout 20° C. Calcium carbonate (0.01 to about 10%, preferably about0.1-2.0%, w/v, based on total volume of the mixture) is added to thecomplexing tank to complex the mixture. Aqueous ammonia or othersuitable base is added while agitating to adjust the pH to a value ofabout 7.0 or greater, preferably about 7.0 to about 9.0, and morepreferably about 7.6 to about 7.8. The temperature is maintained atabout 5 to about 15° C. The pH adjusting base is preferably aqueousammonia having a concentration of about 10% or greater, preferably about20% or greater. Stirring is continued for about 1 hour to obtain asuspension of chlortetracycline calcium complex. After complexing, thecomplexed mixture is pumped to a filter press or other suitable meansfor filtration and separation.

The following Examples refer to treatment of the complexedchlortetracycline mixture obtained in accordance with this teaching toproduce a semi-finished or finished animal feed additive.

EXAMPLE 14 Filtration

The complexed mixture (obtained by either Example 13A or Example 12B) ispumped to a filter press, or other means for separating the liquid, forfiltration. The wet cake is discharged from the filter press andcollected by any convenient means, e.g., into trays, fluid bed, etc.,for drying.

EXAMPLE 15 Drying/Milling

The wet filter cake containing the filtration solids obtained is Example14 is dried at elevated temperature, for example, in an oven by usinghot air flow, a fluid bed, or another suitable drying method asdescribed above. When the moisture content reaches from about 2% toabout 10%, preferably about 4% to about 8%, and most preferably about 4%to about 6%, the drying is ended and the dried product is removed fromthe dryer. In one embodiment, the drying temperature is preferably lessthan about 100° C., preferably from about 60° C. to about 90° C., andmore preferably about 80° C. or less.

The dried product so produced is milled and sized, for example, bygrinding, milling, pulverizing, screening, etc., to produce asemi-finished granular product having a desired particle size. Theparticles are then screened to produce the semi-finished product havingdesired particle size. In one embodiment, the particle size ranges fromabout 180 μm (80 mesh) to about 2 mm (10 mesh). In one embodiment, theparticle size from about 212 μm (70 mesh) to about 1 mm (18 mesh). Inone embodiment, the particles have a size of about 10 mesh or coarser.Particles larger than the desired size may be further milled. Particlessmaller than the desired particle size may be collected and used inother applications, or may be recycled to subsequent batches ofharvested fermentation broth to increase chlortetracycline concentrationin subsequent batches.

The following Examples 9B through 13B illustrate a further embodiment ofthe present invention wherein the chlortetracycline is present in thefree base form.

EXAMPLE 9B Precipitating First Portion of Raw Fermentation Broth

A chlortetracycline-containing fermentation broth is produced andharvested as described in Examples 1-8. The first portion of thefermentation broth is sent to a precipitating tank. The fermentationbroth is optionally cooled, e.g., to below about 20° C. Aqueous ammoniaor other suitable base is added to neutralize the fermentation broth.The base is added in an amount to adjust the pH to a value of about 7.0or greater, preferably from about 7.0 to about 9.0, and more preferablyabout 7.6 to about 7.8. The base is preferably aqueous ammonia having aconcentration of about 10% or greater, preferably about 20% or greater.

EXAMPLE 10B Acidification of Second Portion of Raw Fermentation Broth

The second portion of the harvested broth is sent to an acidificationtank and cooled, preferably from about 15-20° C. The acid used may beany mineral acid or sufficiently strong organic acid as are known tothose skilled in the art. The acid is added in sufficient quantity toadjust the pH to about 4.0 or less, preferably about 3.0 or less, andmost preferably about 2.0 or less. In a preferred embodiment, the acidis an acid that forms a pharmaceutically acceptable salt ofchlortetracycline and most preferably is hydrochloric acid, hydrobromicacid, sulfuric acid, oxalic acid, or mixtures thereof. In an exemplaryembodiment, oxalic acid (2.0% calculated based on total quantity of theharvested broth), is added while agitating and HCl is added to adjustthe pH to about 1.1 to 1.3. Sodium ferrocyanide may be added in amountless than about 2.0% calculated based on total quantity of the harvestedbroth. In an exemplary embodiment, sodium ferrocyanide is added inamount of from about 0.1-0.2%, calculated based on total quantity of theharvested broth. Zinc sulfate may also be added in amount less thanabout 2.0% calculated based on total quantity of the harvested broth. Inan exemplary embodiment, zinc sulfate is added in amount of from about0.1-0.2%, calculated based on total quantity of the harvested broth. Anaqueous solution of oxalic acid (pH<about 3.0, preferably from about 1.5to about 1.8) is then added to dilute the acidified broth to obtainpotency at about 10,000 unit/ml.

EXAMPLE 11B Filtration of Acidified Fermentation Broth

The acidified broth from Example 10B is pumped to a filter press orother means for separating the liquids. The filtrate containing thechlortetracycline hydrochloride is collected and may be sent directlyfor precipitation or, optionally, may be and recirculated through thefilter press for a second filtration prior to being passed on forprecipitation.

EXAMPLE 12B Precipitation of Acidified Filtrate

The acidified filtrate is precipitated in a manner similar to the firstportion. Aqueous ammonia or other suitable base is added to yieldchlortetracycline in the free base form. The base is added to the tankcontaining the acidified broth while agitating to adjust the pH to avalue of about 7.0 or greater, preferably about 7.0 to about 9.0, andmore preferably about 7.6 to about 7.8. The temperature is maintained atabout 5 to about 15° C. The base is preferably aqueous ammonia having aconcentration of about 10% or greater, preferably about 20% or greater.Stirring is continued for about 1 hour to obtain a suspension ofchlortetracycline in the free base form.

EXAMPLE 13B Mixing

The precipitated fermentation broth obtained in Example 9B and thechlortetracycline suspension obtained in Example 12B are mixed and themixture is cooled to about 20° C. or less. The mixture is stirred untilwell mixed, e.g., for about 3 to about 30 minutes, preferably for about5 to about 10 minutes. After mixing, the mixture is pumped to a filterpress or other suitable means for filtration and separation.

The following EXAMPLES 8C through 12C illustrate yet another embodimentof the present invention wherein the feed supplements containchlortetracycline in its free base form.

EXAMPLE 8C Harvesting of Fermentation Broth

A chlortetracycline producing fermentation operation is performed asdescribed in Examples 1-7, above, and the fermentation broth isharvested and divided into two portions as described above in Example8A.

EXAMPLE 9C Acidification of Second Portion of Raw Fermentation Broth

The second portion of the harvested broth (from Example 8C) is acidifiedas described in Example 9A, above.

EXAMPLE 10C Filtration of Acidified Fermentation Broth

The acidified broth from Example 9C is filtered and the filtratecollected as described in Example 10A, above.

EXAMPLE 11C Mixing

The first portion of the raw fermentation broth (from Example 8C) andthe acidified and filtered broth containing chlortetracycline obtainedfrom the second portion (from Example 10C) are mixed, and the mixture isoptionally cooled to about 20° C. or less. The mixture is stirred untilwell mixed, e.g., for about 3 to about 30 minutes, preferably for about5 to about 10 minutes.

EXAMPLE 12C Precipitating Mixture Containing the First Portion of RawFermentation Broth and Acidified Filtrate

The mixture of raw fermentation broth and acidified filtratefermentation from Example 11C broth is optionally cooled, e.g., to belowabout 20° C. Aqueous ammonia or other suitable base is added toneutralize the mixture and yield chlortetracycline in the free baseform. The mixture while agitating to adjust the pH to a value of about7.0 or greater, preferably about 7.0 to about 9.0, and more preferablyabout 7.6 to about 7.8. The temperature is maintained at about 5 toabout 15° C. The pH adjusting base is preferably aqueous ammonia havinga concentration of about 10% or greater, preferably about 20% orgreater. Stirring is continued for about 1 hour to obtain a suspensionof the free base of chlortetracycline. The mixture is pumped to a filterpress or other suitable means for filtration and separation.

EXAMPLE 14C Filtration

The mixture (obtained by either Example 13B or Example 12C) is pumped toa filter press, or other means for separating the liquid, forfiltration. The wet cake is discharged from the filter press andcollected by any convenient means, e.g., into trays, fluid bed, etc.,for drying.

EXAMPLE 15C Drying/Milling

The wet filter cake containing the filtration solids obtained is Example14C is dried and sized as described above in Example 15.

EXAMPLE 16 Finishing

Samples of the semi-finished product obtained in Example 15 or Example15C are drawn and analyzed for active ingredient content, moisture andash content. The process in accordance with this teaching typicallyproduces a semi-finished chlortetracycline product having achlortetracycline content of from about 5% to about 70%. In oneembodiment, the semi-finished product has a chlortetracyclineconcentration of from about 10% to about 66%. In yet another embodiment,the semi-finished product has a chlortetracycline concentration of fromabout 11% to about 65%. In yet another embodiment, the semi-finishedproduct has a chlortetracycline concentration of from about 13% to about60%. In yet another embodiment, the semi-finished product has achlortetracycline concentration of from about 15% to about 55%. In yetanother embodiment, the semi-finished product has a chlortetracyclineconcentration of from about 17% to about 50%. In yet another embodiment,the semi-finished product has a chlortetracycline concentration of fromabout 19% to about 45%. In yet another embodiment, the semi-finishedproduct has a chlortetracycline concentration of from about 20% to about45%. In yet another embodiment, the semi-finished product has achlortetracycline concentration of from about 30% to about 44%.

The semi-finished product may then be standardized to a desired potencyby adding one or more of an edible feed material, a mineral product, andan oil in ratios of a sufficient quantity to produce the desiredpotency. In admixing the ingredients, the solid ingredients may beadmixed, for example, with a feed blender, by hand, and so forth. In oneembodiment, the semi-finished product and edible feed material areplaced in a blender and the oil is sprayed over the carriers during themixing process.

The edible feed material, mineral product, semi-finished product, andoil are preferably combined in the following concentrations by weight:

Ingredient Concentration Edible feed material 0.0 to 90% Mineral product0.0 to 89% Semi-finished product 2.4 to 98% Oil or fat 0.0 to 15%

In a preferred embodiment, the raw material is assayed and thequantities of edible feed material and mineral product are adjusted toprovide a standardized concentration or potency of the antibiotic.

Suitable edible feed materials include chaffs, silage, meals, pellets,concentrates or the like formed from grains, nuts, beans and other plantparts, including for example, grain products such as corn, rice, wheat,milo, rice bran, other straws or grasses rich in crude fibrous material,ground corn cobs, oil seeds and by products, soybean mill run, wheatmiddlings, wheat bran oat groats, oat bran, barley, alfalfa meal chaffor silage, wheat germ, corn germ meal, soybean grits, corn gluten feed,soybean meal, coconut meal, soya bean meal, peanut meal, corn cob meal,linseed meal, cottonseed meal, alfalfa leaf meal, rice hulls, and thelike. Other edible feed materials include dried beet pulp, driedmolasses beet pulp, dried potato pulp, dried citrus pulp, dried brewersgrains, corn distillers dried grains, oat mill feed, heat treated soymill feed, etc. The edible feed material is preferably rice hull.

If an oil is to be used in preparing the finished feed supplementsaccording to the present invention, any non-toxic oil or fat may be usedin the formulation. Suitable oils include, for example, mineral oil,waxes, cotton seed oil, palm oil, peanut oil, corn oil, sesame oil,coconut oil, soybean oil, grapeseed oil, linseed oil, lard, tallow, andthe like. The oil is preferably mineral oil. It is not necessary thatoil, if it is to be added, be added during the finishing stage and maybe added earlier in the process, such as during the fermentation stage.The oil may be used during fermentation as an assimilable source ofcarbon and thus, some of the excess oil may be carried over into thesemi-finished product.

The mineral product may be one or more mineral products suitable for usein an animal feed or feed supplement or other veterinary use. Exemplarymineral products include but are not limited to ammonium sulfate, animalbone ash, animal bone charcoal, animal bone charcoal (spent), animalbone meal (cooked or boiled), animal bone meal (steamed), animal bonephosphate, calcite (ground), calcium carbonate, calcium carbonate(precipitated), calcium chloride, calcium formate, calcium gluconate,calcium hydroxide, calcium iodate, calcium iodobehenate, calcium oxide,calcium periodate, calcium sulfate, chalk (precipitated), chalk rock(ground), clam shells (ground), cobalt acetate, cobalt carbonate, cobaltchloride, cobalt choline citrate complex, cobalt glucoheptonate, cobaltgluconate, cobalt oxide, cobalt sulfate, copper carbonate, cuprouschloride, cupric chloride, copper choline citrate complex, cupricgluconate, cupric hydroxide, cupric orthophosphate, cuprous oxide,cupric oxide, cupric sulfate, cuprous iodide, diiodosalicylic acid,ethylene diamine dihydroiodide, ferric ammonium citrate, ferricchloride, ferric choline citrate complex, ferric formate, ferricphosphate, ferric pyrophosphate, ferric sulfate, ferrous carbonate,ferrous chloride, ferrous fumarate, ferrous gluconate, ferrous glycinecomplex, ferrous sulfate, gypsiferrous shale, ferric oxide, reducediron, limestone (ground), limestone (dolomitic ground), limestone(magnesium ground), magnesium carbonate, magnesium chloride, magnesiumhydroxide, magnesium phosphate, magnesium oxide, magnesium-mica,magnesium sulfate, manganese acetate, manganous carbonate, manganouschloride, manganous citrate, manganous gluconate, manganeseorthophosphate, manganese phosphate dibasic, manganous sulfate,manganous oxide, metal amino acid complexes, copper amino acid complex,zinc amino acid complex, magnesium amino acid complex, iron amino acidcomplex, cobalt amino acid complex, calcium amino acid complex, calciumamino acid complex, potassium amino acid complex, manganese amino acidcomplex, copper lysine complex, zinc lysine complex, ferric methioninecomplex, manganese methionine complex, zinc methionine complex, metalamino acid chelates, calcium amino acid chelate, cobalt amino acidchelate, copper amino acid chelate, iron amino acid chelate, magnesiumamino acid chelate, manganese amino acid chelate, zinc amino acidchelate, metal polysaccharide complex, copper polysaccharide complex,iron polysaccharide complex, zinc polysaccharide complex, magnesiumpolysaccharide complex, metal proteinate, copper proteinate, zincproteinate, magnesium proteinate, iron proteinate, cobalt proteinate,manganese proteinate, calcium proteinate, oyster shell flour, ammoniumpolyphosphate solution, calcium phosphate, diammonium phosphate,dicalcium phosphate, disodium phosphate, monoammonium phosphate,monocalcium phosphate, monosodium phosphate, phosphoric acid,defluorinated phosphate, rock phosphate (soft), rock phosphate (ground),rock phosphate (ground, low fluorine), sodium hexametaphosphate, sodiumtripolyphosphate, tribasic sodium phosphate, tricalcium phosphate,potassium bicarbonate, potassium carbonate, potassium citrate, potassiumchloride, potassium hydroxide, potassium iodate, potassium iodide,potassium sulfate, sodium chloride, sodium chloride (iodized), shellflour, sodium acid pyrophosphate, sodium bicarbonate, sodium carbonate,sodium iodate, sodium iodide, sodium molybdate, sodium selenate, sodiumselenite, sodium sesquicarbonate, sodium sulfate, sulfur, thymol iodide,zinc acetate, zinc carbonate, zinc chloride, zinc chloride diamminecomplex, zinc oxide, zinc sulfate, bone phosphate, copper acetate, basiccopper chloride (Cu₂(OH)₃Cl), and chromium picolinate.

In one embodiment, the mineral product is limestone. By “limestone” ismeant not only naturally occurring form calcium carbonate such chalk,dolomite, etc., but also is intended to encompass veterinary,agricultural, and technical grade calcium carbonate. Of course, highergrades of calcium carbonate may be employed as well.

In one embodiment, where an edible feed material and/or mineral productare utilized, they are also granular in nature and are sized so as to besubstantially the same size as the particles of the semi-finishedproduct, thereby minimizing their separation during handling.

In one embodiment, the resulting composition has a chlortetracyclineconcentration of from about 5% to about 70%. In another embodiment, theresulting composition has a chlortetracycline concentration of fromabout 10% to about 66%. In yet another embodiment, the resultingcomposition has a chlortetracycline concentration of from about 11% toabout 65%. In yet another embodiment, the resulting composition has achlortetracycline concentration of from about 13% to about 60%. In yetanother embodiment, the resulting composition has a chlortetracyclineconcentration of from about 15% to about 55%. In yet another embodiment,the resulting composition has a chlortetracycline concentration of fromabout 17% to about 50%. In yet another embodiment, the resultingcomposition has a chlortetracycline concentration of from about 19% toabout 45%. In yet another embodiment, the resulting composition has achlortetracycline concentration of from about 20% to about 45%. In yetanother embodiment, the resulting composition has a chlortetracyclineconcentration of from about 30% to about 44%. In some of the presentlypreferred embodiments, the animal feed supplement in accordance withthis teaching provides chlortetracycline calcium complex in an amounthaving an activity equivalent to 50, 60, 70, 80, 90, 100, or 200 gramsof chlortetracycline hydrochloride per pound of animal feed supplement.

The animal feed premixes according to the present invention may alsocontain additional ingredients as typically used in animal husbandry,such as coccidiostats, additional antibiotics, minerals, vitamins,growth promoters, antioxidants, anthelmintics, preservatives, colorings,flavorings, or other dietary supplements.

In order to provide for comprehension of the invention, for illustrativepurposes and without a restrictive scope, a description is furnished asto the details of particular embodiments of the invention, makingreference to FIGS. 5 and 6, in which diagramed arrangements of twoexemplary systems designed for carrying out the processes in accordancewith the present invention are shown.

As is shown in FIG. 5, the system comprises a fermentor 10 for producingchlortetracycline as is generally known in the art. Following thefermentation process, it is shown in the drawing that the fermentationbroth is discharged from fermentor 10 so that a first portion of theharvested fermentation broth is routed directly to mixing tank 24 forprecipitation of the chlortetracycline (either as the free base or as acomplex) and a second portion is routed to acidification tank 12 foracidification. Alternatively, as stated above, rather than dividing asingle fermentation batch into two portions, the two portions may beharvested from separate fermentors (see FIG. 6) or separate fermentationbatches from the same fermentor. The acidified portion is pumped viapump 14 to filter press 16 and the filtrate is collected in filtratepool 18. The filtrate may optionally be recirculated to filter 16 forone or more additional filtrations and the filtrate is pumped via pump20 to precipitation tank 22 for precipitation, either as the free base,or as a complex. The precipitated filtrate is then passed on to mixingtank 24 for admixture with the previously treated fermentation broth. Ifthe acidified filtration broth is filtered only once, filtrate pool 18may be omitted and the filtrate may be passed on directly from filterpress 16 to tank 22. The combined mixture is then pumped via pump 26through filter press 28. The wet filter cake is removed from filterpress 28 and is transported by whatever means are convenient, such astrolley 30, to dryer 32 to produce the dry product then to anappropriate milling or sizing apparatus 34 for preparation of thesemi-finished product.

The circuit, as shown in FIG. 5, comprises a plurality of like orsimilar devices, e.g., a plurality of filter presses, mixing tanks, andpumps. Thus, in an alternative embodiment, this device affords thepossibility of replacing a plurality of like or similar devices with asingle unit by alternating its use.

Referring now to FIG. 6, the system comprises a plurality of fermentors10 and 11 for producing chlortetracycline as is generally known in theart. Following the fermentation process, it is shown in the drawing thatthe fermentation broth is discharged from fermentor 10 and routeddirectly to mixing tank 22 and the fermentation broth from fermentor 11is routed to acidification tank 12 for acidification. Alternatively, asstated above, rather than employing separate fermentors 10 and 11, asingle fermentation batch may be divided into two portions (see FIG. 5),the two fermentation batches may be harvested from separate fermentationbatches produced in the same fermentor. The acidified portion is pumpedvia pump 14 to filter press 16 and the filtrate is collected in filtratepool 18. The filtrate may optionally be recirculated to filter 16 forone or more additional filtrations and the filtrate is pumped via pump20 to precipitation tank 22 and mixed with the raw fermentation brothdischarged from fermentor 10. If the acidified filtration broth isfiltered only once, filtrate pool 18 may be omitted and the filtrate maybe passed on directly from filter press 16 to tank 22. The mixture istreated to precipitate the chlortetracycline, either as the free base,or as a complex and is then pumped via pump 26 through filter press 28.The wet filter cake is removed from filter press 28 and is transportedby whatever means are convenient, such as trolley 30, to dryer 32 toproduce the dry product then to an appropriate milling or sizingapparatus 34 for preparation of the semi-finished product.

The circuit, as shown in FIG. 6, comprises a plurality of like orsimilar devices, e.g., a plurality of fermentors, filter presses, mixingtanks, and pumps. Thus, in an alternative embodiment, this deviceaffords the possibility of replacing a plurality of like or similardevices with a single unit by alternating its use.

The description above should not be construed as limiting the scope ofthe invention, but as merely providing illustrations to some of thepresently preferred embodiments of this invention. In light of the abovedescription and examples, various other modifications and variationswill now become apparent to those skilled in the art without departingfrom the spirit and scope of the present invention as claims.Accordingly, the scope of the invention should be the appended claimsand their legal equivalents. All are incorporated by reference in theirentireties.

1. A method of combating microbial infection in animals comprisingorally administering to said animals a prophylactic or therapeuticamount of an animal comestible composition comprising a medicatedsupplement prepared by culturing an organism producing achlortetracycline in a fermentation medium to produce a fermentationbroth; adding an additional quantity of fermentation solid comprisingsaid chlortetracycline to the fermentation broth to increase theantibiotic activity of said fermentation broth; reducing saidfermentation broth to obtain fermentation solids comprising saidchlortetracycline; drying said fermentation solids to produce a drysolid; granulating said dry solid to produce granulated fermentationsolids comprising uncompacted granules having a substantially uniformparticle size, the granules having an antibiotic concentration in therange of 10 g/lb through 300 g/lb.
 2. The method as described in claim1, further comprising the step of blending said granulated fermentationsolids with at least one potency standardizer.
 3. The method asdescribed in claim 1, further comprising the step of blending saidgranulated fermentation solids with an edible oil.
 4. The method asdescribed in claim 1, wherein the additional quantity of saidchlortetracycline added to the fermentation broth was obtained from aprevious batch of fermentation broth.
 5. The method as described inclaim 1, wherein the granules have an antibiotic concentration of about200 g/lb.
 6. A method of combating microbial infection in animalscomprising orally administering to said animals a prophylactic ortherapeutic amount of an animal comestible composition comprising amedicated supplement prepared by culturing an organism producing achlortetracycline in a first fermentation medium to produce afermentation broth; reducing said fermentation broth to obtainfermentation solids comprising said chlortetracycline; drying saidfermentation solids to produce a dry solid; granulating said dry solidto produce granulated fermentation solids; screening the granulatedfermentation solids to arrive at a first group of granulated solidscorresponding to a desired mesh size, the granules having an antibioticconcentration in the range of 10 g/lb through 300 g/lb and a secondgroup of solids which do not correspond with the desired mesh size; andadding the second group of solids to a second, subsequent, fermentationbroth having an organism producing chlortetracycline in a secondfermentation medium for increasing the antimicrobial concentration of asubsequent animal comestible composition, for combating microbialinfection in animals.
 7. The method as described in claim 6, furthercomprising: reducing said second fermentation broth to obtain a thirdgroup of fermentation solids comprising said chlortetracycline, dryingsaid third group of fermentation solids to produce a dry solid; andgranulating said dry solid to produce granulated fermentation solids. 8.The method as described in claim 7, wherein the granules have anantibiotic concentration of about 200 g/lb.